Methods for producing citric acid using host cells deficient in oxaloacetate hydrolase

ABSTRACT

The present invention relates to isolated nucleic acid sequences encoding polypeptides having oxaloacetate hydrolase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/336,491filed Jan. 3, 2003, now U.S. Pat. No. 6,936,438, which is a division ofU.S. application Ser. No. 09/501,612 filed Feb. 10, 2000, now U.S. Pat.No. 6,544,765, which claims priority or the benefit under 35 U.S.C. 119of Danish application no. PA 1999 00231 filed Feb. 22, 1999 and U.S.provisional application Ser. No. 60/121,481 filed Feb. 24, 1999, thecontents of which are fully incorporated herun by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to isolated nucleic acid sequences encodingpolypeptides having oxaloacetate hydrolase activity. The presentinvention further relates to mutant host cells, in particular fungalmutant host cells such as cells of the genus Aspergillus, deficient inoxaloacetate hydrolase activity and thereby in oxalic acid production.The present invention also relates to the use of the mutant cells forproducing desirable compounds, such as polypeptides, primary andsecondary metabolites, and to methods of producing such compounds in themutant cells of the invention. The invention also relates to nucleicacid constructs, vectors, and host cells comprising the nucleic acidsequences as well as recombinant methods for producing the polypeptideshaving oxaloacetate hydrolase activity.

2. Description of Related Art

Filamentous fungi are widely used for the commercial production of avariety of compounds of interest, including homologous compounds, suchas primary or secondary metabolites and polypeptides normally producedby the fungus in question or heterologous compounds, such asheterologous polypeptides encoded by foreign DNA which has beenintroduced into the fungus in question. Such products are produced byfermentation of the fungus in question and harvest of the desiredproduct resulting from the fermentation.

The fungal species A. niger is widely used for the commercial productionof desired compounds, e.g., citric acid and industrial enzymes. It iswell-known that this species produces large amounts of oxalic acid. Fora number of reasons the production of oxalic acid is undesirably whenthis species is used for commercial production of a compound ofinterest. For instance, the production of oxalic acid requires a lot ofcarbon and thus extra, expensive carbon sources must be added to thefermentation medium compared to what would be required only for theproduction of the desired compound; the presence of oxalic acid in thefermentation broth causes problems in the downstream processing involvedin the recovery of the product of interest because oxalic acid forms aprecipitate with calcium which interferes in the recovery; and oxalicacid is a toxic compound which means that its presence is considered amajor problem in the production of food grade products from A. niger.

Two possible routes for the pathway for biosynthesis of oxalic acid inA. niger have been suggested. The first route isoxaloacetate+water→Oxalic acid+acetate, the reaction being catalyzed byoxaloacetate hydrolase (Kubicek, C. P., G. Schreferl-Kunar, W. Wöhrerand M. Röhr, Appl. Environ. Microbiol. 54: 633–637 (1988)). The secondroute involves the glycoxylate pathway (Balmforth, A. J., A. Thomson,Biochem. J. 218: 113–118 (1984)).

It has been attempted to control the formation of oxalic acid duringcommercial A. niger fermentations by conducting the fermentation at alow pH where only little oxalic acid is formed. However, fermentation atlow pH may be undesirable since normally this pH is not optimal for thegrowth of A. niger and yield of a desired fermentation product.

A partially purified oxaloacetate hydrolase from A. niger has beendescribed (Lenz et al., Partial purification and some properties ofoxaloacetate from Aspergillus niger, Eur. J. Biochem. 65: 225–236(1976)), but the gene encoding this enzyme has not been described.

It is an object of the present invention to provide mutants of cells,such as filamentous fungal cells, in particular cells of A. niger, whichis deficient in oxaloacetate hydrolase production and thereby oxalicacid production.

SUMMARY OF THE INVENTION

The present invention relates to isolated nucleic acid sequencesencoding polypeptides having oxaloacetate hydrolase activity, selectedfrom the group consisting of:

(a) a nucleic acid sequence encoding a polypeptide having an amino acidsequence which has at least 65% identity with the amino acid sequence ofthe oxaloacetate hydrolase of SEQ ID NO: 2;

(b) a nucleic acid sequence having at least 65% homology with the codingpart of the DNA sequence SEQ ID NO: 1 (constituted by nucleotides1157–1411, 1504–1651 and 1764–2383 of SEQ ID NO: 1);

(c) a nucleic acid sequence which hybridizes under medium stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 1, (ii) thecDNA sequence of SEQ ID NO: 1, (iii) a subsequence of (i) or (ii) of atleast 100 nucleotides, or (iv) a complementary strand of (i), (ii), or(iii);

(d) an allelic variant of (a), (b), or (c); and

(e) a subsequence of (a), (b), (c), or (d), wherein the subsequenceencodes a polypeptide fragment which has oxaloacetate hydrolaseactivity.

In a further important aspect the invention relates to a method forproducing a mutant of a cell, which comprises disrupting or deleting thenucleic acid sequence of the invention or a control sequence thereof,which results in the mutant producing less of the oxaloacetate hydrolaseand thus oxalic acid than the cell. The present invention also relatesto a mutant produced by this method.

The present invention also relates to nucleic acid constructs, vectors,and host cells comprising the nucleic acid sequences as well asrecombinant methods for producing the polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the restriction map of Aspergillus niger pyrG plasmidpJRoy10.

FIG. 2 shows the construction of plasmid pHP3 described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

Isolated Nucleic Acid Sequences Encoding Polypeptides HavingOxaloacetate Hydrolase Activity

The term “oxaloacetate hydrolase activity” is defined herein as anactivity which catalyzes the reaction: oxaloacetate+water→Oxalicacid+acetate. The enzyme is classified as belonging to EC 3.7.1.1. Forpurposes of the present invention, oxaloacetate hydrolase activity isdetermined according to the procedure described in the Materials andMethods section further below. One unit of oxaloacetate hydrolaseactivity is defined as 1.0 μmole of oxalic acid produced per minute at30° C., pH 7.5.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

In a first embodiment, the present invention relates to isolated nucleicacid sequences encoding polypeptides having an amino acid sequence whichhas a degree of identity to amino acids 1 to 341 of SEQ ID NO: 2 (i.e.,the mature polypeptide) of at least about 65%, preferably at least about70%, more preferably at least about 80%, even more preferably at leastabout 90%, most preferably at least about 95%, and even most preferablyat least about 97%, which have oxaloacetate hydrolase activity(hereinafter “homologous polypeptides”). In a preferred embodiment, thehomologous polypeptides have an amino acid sequence which differs byfive amino acids, preferably by four amino acids, more preferably bythree amino acids, even more preferably by two amino acids, and mostpreferably by one amino acid from amino acids 1 to 341 of SEQ ID NO: 2.For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,CABIOS 5: 151–153 (1989)) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10, and gap length penaltyof 10. Pairwise alignment parameters were Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

Preferably, the nucleic acid sequences of the present invention encodepolypeptides that comprise or consist of the amino acid sequence of SEQID NO: 2 or an allelic variant thereof; or a fragment thereof that hasoxaloacetate hydrolase activity.

The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the amino acid sequence of SEQ ID NO: 2,which differ from SEQ ID NO: 1 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO: 1 which encode fragments of SEQ ID NO: 2 which have oxaloacetatehydrolase activity or which are sufficiently long to be used forinactivation of an oxaloacetate hydrolase gene in a microbial cell (asdescribed in the section entitled “Removal or Reduction of OxaloacetateHydrolase Activity”).

A subsequence of SEQ ID NO: 1 is a nucleic acid sequence encompassed bySEQ ID NO: 1 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Preferably, a subsequence contains at least 2800nucleotides, more preferably at least 3000 nucleotides, and mostpreferably at least 3200 nucleotides. A fragment of SEQ ID NO: 2 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxy terminus of this amino acid sequence. Preferably, a fragmentcontains at least 270 amino acid residues, more preferably at least 300amino acid residues, and most preferably at least 320 amino acidresidues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chomosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. The allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The amino acid sequences of the homologous polypeptides may differ fromthe amino acid sequence of SEQ ID NO: 2 by an insertion or deletion ofone or more amino acid residues and/or the substitution of one or moreamino acid residues by different amino acid residues. Preferably, aminoacid changes are of a minor nature, that is conservative amino acidsubstitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20–25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (such as arginine, lysine and histidine), acidic amino acids(such as glutamic acid and aspartic acid), polar amino acids (such asglutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine and valine), aromatic amino acids (such as phenylalanine,tryptophan and tyrosine), and small amino acids (such as glycine,alanine, serine, threonine and methionine). Amino acid substitutionswhich do not generally alter the specific activity are known in the artand are described, for example, by H. Neurath and R. L. Hill, 1979, In,The Proteins, Academic Press, New York. The most commonly occurringexchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.

In a second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to the maturepolypeptide coding sequence of SEQ ID NO: 1 (constituted by nucleotides1157–1411, 1504–1651 and 1764–2383 of SEQ ID NO: 1) of at least about65%, preferably about 70%, preferably about 80%, more preferably about90%, even more preferably about 95%, and most preferably about 97%homology, which encode an active polypeptide or which are sufficientlylong to be used for inactivation of an oxaloacetate hydrolase gene in amicrobial cell (as described in the section entitled “Removal orReduction of Oxalolacetate Hydrolase Activity”); or allelic variants andsubsequences of SEQ ID NO: 1 which encode polypeptide fragments whichhave oxaloacetate hydrolase activity or which are sufficiently long tobe used for inactivation of an oxaloacetate hydrolase gene in amicrobial cell (as described in the section entitled “Removal orReduction of Oxaloacetate Hydrolase Activity”). For purposes of thepresent invention, the degree of homology between two nucleic acidsequences is determined by the computer program Align provided in theFasta program package (Version v20u6), using GAP with the followingsettings for nucleotide sequence comparison: GAP creation penalty (forthe first residue) in a GAP of −16 and GAP extension (for the additionalresidues) penalty of −4. Align is a slightly modified version of aprogram taken from E. Myers and W. Miller. The algorithm is described inE. Myers and W. Miller, “Optimal Alignments in Linear Space” (CABIOS 4:11–17 (1988)).

In a third embodiment, the present invention relates to isolated nucleicacid sequences encoding polypeptides having oxaloacetate hydrolaseactivity which hybridize under medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with a nucleic acid probe which hybridizes under the sameconditions with (i) the nucleic acid sequence of SEQ ID NO: 1, (ii) thecDNA sequence of SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or(iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F.Fritsch, and T. Maniatus, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y. (1988)). The subsequence of SEQ ID NO:1 may be at least 100 nucleotides or preferably at least 200nucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has oxaloacetate hydrolase activity.

The nucleic acid sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having oxaloacetate hydrolase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 35 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having oxaloacetate hydrolase activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1 or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleic acid sequencehybridizes to a nucleic acid probe corresponding to the nucleic acidsequence shown in SEQ ID NO: 1, its complementary strand, or asubsequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred embodiment, the nucleic acid probe is SEQID NO: 1. In another preferred embodiment, the nucleic acid probe is thenucleotides encoding the fragment of SEQ ID NO: 2 constituted by aminoacid residues 123–205 or a part of this fragment. In another preferredembodiment, the nucleic acid probe is the nucleic acid sequencecontained in plasmid pHP1 which is contained in Escherichia coliDSM-12660 wherein the nucleic acid sequence encodes a polypeptide havingoxaloacetate hydrolase activity, in particular the mature polypeptidecoding region of SEQ ID NO: 1.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (Proceedings of the National Academy of Sciences U.S.A., 48:1390 (1962)) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5%NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodiummonobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under medium, medium-high, high, orvery high stringency conditions with the sequence of SEQ ID NO: 1, orits complementary strand, or a subsequence thereof; and (b) isolatingthe nucleic acid sequence. The subsequence is preferably a sequence ofat least 100 nucleotides such as a sequence which encodes a polypeptidefragment which has oxaloacetate hydrolase activity or a sequence whichis sufficiently long to be used for inactivation of an oxaloacetatehydrolase gene in a microbial cell (as described in the section entitled“Removal or Reduction of Oxaloacetate Hydrolase Activity”).

The polypeptides encoded by the isolated nucleic acid sequences of thepresent invention have at least 20%, preferably at least 40%, morepreferably at least 60%, even more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 100% of theoxaloacetate hydrolase activity of the mature polypeptide of SEQ ID NO:2.

The nucleic acid sequences of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted.

The nucleic acid sequences may be obtained from a bacterial source. Forexample, these polypeptides may be obtained from a gram positivebacterium such as a Bacillus strain, e.g., Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus stearothermophilus,Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain,e.g., Streptomyces lividans or Streptomyces murinus; or from a gramnegative bacterium, e.g., E. coli or Pseudomonas sp.

In a preferred embodiment, the nucleic acid sequence is obtained from afungal cell. “Fungi” as used herein includes the phyla Ascomycota,Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,CAB International, University Press, Cambridge, UK (1995)) as well asthe Oomycota (as cited in Hawksworth et al., supra, page 171 (1995)) andall mitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal cell is a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, (1980)).

More specifically, the nucleic acid sequences may be obtained from ayeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia strain; or morepreferably from a filamentous fungal strain such as an Acremonium,Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermastrain.

In a preferred embodiment, the nucleic acid sequences are obtained froma Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis strain.

In another preferred embodiment, the nucleic acid sequences are obtainedfrom an Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride strain.

In a more preferred embodiment, the nucleic acid sequences are obtainedfrom Aspergillus niger, and most preferably from A. niger BO1 DSM 12665,e.g., the nucleic acid sequence set forth in SEQ ID NO: 1. In anothermore preferred embodiment, the nucleic acid sequence is the sequencecontained in plasmid pHP1 which is contained in Escherichia coliDSM-12660. In another preferred embodiment, the nucleic acid sequence isa nucleotide sequence constituted by nucleotides 1157–1411, 1504–1651and 1764–2383 of SEQ ID NO: 1, which encodes the mature polypeptide ofSEQ ID NO: 2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents. For example, the polypeptides maybe obtained from microorganisms which are taxonomic equivalents ofAspergillus niger as defined by Benn and Klich (Benn J. W. and M. A.Klich, Aspergillus, Biology and industrial applications.Butterworth-Heinemann, U.S.A. (1992)), regardless of the species name bywhich they are known, e.g., A. aculeatus, A. awamori, A. carboniarus, A.ellipticus, A. ficuum, A. foetidus, A. heteromorphus, A. japonicus, A.phoenicis, A. pulverulentus, A. tubingensis, A. helicothrix, A.atroviolaceus, A. citricus, A. acidicus or A. fonsecaeus.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such nucleic acid sequences may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al., PCR:A Guide to Methods and Application, Academic Press, New York (1990). ForPCR it may be of particular relevance to use a set of primers spanningthe nucleotide sequence encoding amino acids 123–205 of SEQ ID NO: 2.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Aspergillus, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

Modification of a nucleic acid sequence of the present invention may benecessary for the synthesis of polypeptides substantially similar to thepolypeptide. The term “substantially similar” to the polypeptide refersto non-naturally occurring forms of the polypeptide. These polypeptidesmay differ in some engineered way from the polypeptide isolated from itsnative source, e.g., variants that differ in specific activity,thermostability, pH optimum, or the like. The variant sequence may beconstructed on the basis of the nucleic acid sequence presented as thepolypeptide encoding part of SEQ ID NO: 1, e.g., a subsequence thereof,and/or by introduction of nucleotide substitutions which do not giverise to another amino acid sequence of the polypeptide encoded by thenucleic acid sequence, but which corresponds to the codon usage of thehost organism intended for production of the enzyme, or by introductionof nucleotide substitutions which may give rise to a different aminoacid sequence. For a general description of nucleotide substitution,see, e.g., Ford et al., Protein Expression and Purification 2: 95–107(1991).

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, Science 244: 1081–1085 (1989)). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor oxaloacetate hydrolase activity to identify amino acid residues thatare critical to the activity of the molecule. Sites of substrate-enzymeinteraction can also be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labelling (see, e.g., de Voset al., Science 255: 306–312 (1992); Smith et al., Journal of MolecularBiology 224: 899–904 (1992); Wlodaver et al., FEBS Letters 309: 59–64(1992)).

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutantnucleic acid sequence, comprising introducing at least one mutation intothe mature polypeptide coding sequence of SEQ ID NO: 1 or a subsequencethereof, wherein the mutant nucleic acid sequence encodes a polypeptidewhich consists of amino acids 1 to 341 of SEQ ID NO: 2 or a fragmentthereof which has oxaloacetate hydrolase activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

Removal or Reduction of Oxaloacetate Hydrolase Activity

The present invention also relates to methods for producing a mutantcell of a parent cell, which comprises disrupting or deleting a nucleicacid sequence of the present invention or a control sequence thereof,which results in the mutant cell producing less of the polypeptidehaving oxaloacetate hydrolase activity encoded by the nucleic acidsequence than the parent cell and thus less oxalic acid when cultivatedunder the same conditions.

The construction of strains that have reduced oxaloacetate hydrolaseactivity and oxalic acid production may be conveniently accomplished bymodification or inactivation of a nucleic acid sequence necessary forexpression of the polypeptide having oxaloacetate hydrolase activity inthe cell. The nucleic acid sequence to be modified or inactivated maybe, for example, a nucleic acid sequence encoding the polypeptide or apart thereof essential for exhibiting oxaloacetate hydrolase activity,e.g., a nucleic acid sequence of the invention as described in thesection entitled “Isolated Nucleic Acid Sequences Encoding PolypeptidesHaving Oxaloactate Hydrolase Activity”, or the nucleic acid sequence mayhave a regulatory function required for the expression of thepolypeptide from the coding sequence of the nucleic acid sequence. Anexample of such a regulatory or control sequence may be a promotersequence or a functional part thereof, i.e., a part which is sufficientfor affecting expression of the polypeptide. Other control sequences forpossible modification are described further in this section.

Modification or inactivation of the nucleic acid sequence may beperformed by subjecting the cell to mutagenesis and selecting orscreening for cells in which the oxaloacetate hydrolase producingcapability has been reduced. The mutagenesis, which may be specific orrandom, may be performed, for example, by use of a suitable physical orchemical mutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor cells exhibiting reduced oxaloacetate hydrolase activity orproduction. The reduced or eliminated oxaloacetate hydrolase activitymay be determined by use of the assay described in the Examples below.

Modification or inactivation of production of a polypeptide encoded by anucleic acid sequence of the present invention may be accomplished byintroduction, substitution, or removal of one or more nucleotides in thenucleic acid sequence encoding the polypeptide or a regulatory elementrequired for the transcription or translation thereof. For example,nucleotides may be inserted or removed so as to result in theintroduction of a stop codon, the removal of the start codon, or achange of the open reading frame. Such modification or inactivation maybe accomplished by site-directed mutagenesis or PCR generatedmutagenesis in accordance with methods known in the art. Although, inprinciple, the modification may be performed in vivo, i.e., directly onthe cell expressing the nucleic acid sequence to be modified, it ispreferred that the modification be performed in vitro as exemplifiedbelow.

An example of a convenient way to eliminate or reduce production by ahost cell of choice is based on techniques of gene replacement or geneinterruption. For example, in the gene interruption method, a nucleicacid sequence corresponding to the endogenous gene or gene fragment ofinterest is mutagenized in vitro to produce a defective nucleic acidsequence which is then transformed into the host cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous gene or gene fragment. It may bedesirable that the defective gene or gene fragment also encodes a markerwhich may be used for selection of transformants in which the geneencoding the polypeptide has been modified or destroyed.

Alternatively, modification or inactivation of the nucleic acid sequencemay be performed by established anti-sense techniques using a nucleotidesequence complementary to the polypeptide encoding sequence. Morespecifically, production of the polypeptide by a cell may be reduced oreliminated by introducing a nucleotide sequence complementary to thenucleic acid sequence encoding the polypeptide which may be transcribedin the cell and is capable of hybridizing to the polypeptide mRNAproduced in the cell. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the polypeptide mRNA, theamount of polypeptide translated is thus reduced or eliminated.

It is preferred that the cell to be modified in accordance with themethods of the present invention is of microbial origin, in particularof any of the species listed in the section above entitled “IsolatedNucleic Acid Sequences Encoding Polypeptides Having OxaloacetateHydrolase Activity” as sources for the nucleic acid sequence of theinvention encoding a polypeptide with oxaloacetate hydrolase activity.Preferably, the cell is a fungal strain which is suitable for theproduction of desired products such as polypeptides or primary orsecondary metabolites, either homologous or heterologous to the cell.Even more preferably, the cell is a cell of Aspergillus, in particularA. niger.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleic acid sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide than the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous expression products, such as homologous or heterologouspolypeptides or primary or secondary metabolites. Therefore, the presentinvention further relates to methods for producing a homologous orheterologous product comprising (a) cultivating the mutant cell underconditions conducive for production of the product; and (b) recoveringthe product.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art, e.g., asdescribed further below in the section entitled “Methods of Production”.

The methods of the present invention for producing an essentially oxalicacid free product is of particular interest in the production of primarymetabolites, in particular food grade products such as citric acid andother Krebs cyclus acids, and homologous or heterologous polypeptides,in particular eukaryotic polypeptides.

The polypeptide may be any polypeptide heterologous or homologous to themutant cell. The term “polypeptide” is not meant herein to refer to aspecific length of the encoded product, and therefore, encompassespeptides, oligopeptides, and proteins. The term “heterologouspolypeptide” is defined herein as a polypeptide that is not native tothe mutant cell, a native (or protein in which modifications have beenmade to alter the native sequence, or a native protein whose expressionis quantitatively altered as a result of a manipulation of the mutantcell by recombinant DNA techniques. The mutant cell may contain one ormore copies of the nucleic acid sequence encoding the homologous orheterologous polypeptide. In a preferred embodiment, the heterologouspolypeptide is an extracellularly secreted polypeptide.

Preferably, the polypeptide to be produced is a hormone, hormonevariant, enzyme, receptor or portion thereof, antibody or portionthereof, or reporter. The enzyme may be selected from, e.g., anamylolytic enzyme, lipolytic enzyme, proteolytic enzyme, cellulyticenzyme, oxidoreductase, or plant cell-wall degrading enzyme. Examples ofsuch enzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,galactosidase, beta-galactosidase, glucoamylase, glucose oxidase,glucosidase, haloperoxidase, hemicellulase, invertase, isomerase,laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolyticenzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transferase, transglutaminase, orxylanase. The oxaloacetate hydrolase-deficient cells may also be used toexpress heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like.

The nucleic acid sequence encoding a heterologous polypeptide that canbe expressed in a mutant cell of the invention, in particular afilamentous fungal mutant cell, may be obtained from any prokaryotic,eukaryotic, or other source. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide is produced by the source or by a cellin which a gene from the source has been inserted.

In the methods of the present invention, the mutant cell, in particulara mutant filamentous fungal cell may also be used for the recombinantproduction of polypeptides or other products such as primary orsecondary metabolites that are native (or “homologous”) to the cell.

The techniques used to isolate or clone a nucleic acid sequence encodinga heterologous polypeptide are known in the art and include isolationfrom genomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the nucleic acid sequence from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR).See, for example, Innis et al., PCR Protocols: A Guide to Methods andApplication, Academic Press, New York (1990). The cloning procedures mayinvolve excision and isolation of a desired nucleic acid fragmentcomprising the nucleic acid sequence encoding the polypeptide, insertionof the fragment into a vector molecule, and incorporation of therecombinant vector into the mutant cell where multiple copies or clonesof the nucleic acid sequence will be replicated. The nucleic acidsequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin,or any combinations thereof.

In the methods of the present invention, heterologous polypeptides mayalso include fused or hybrid polypeptides in which another polypeptideis fused at the N-terminus or the C-terminus of the polypeptide orfragment thereof. A fused polypeptide is produced by fusing a nucleicacid sequence (or a portion thereof) encoding one polypeptide to anucleic acid sequence (or a portion thereof) encoding anotherpolypeptide. Techniques for producing fusion polypeptides are known inthe art, and include, ligating the coding sequences encoding thepolypeptides so that they are in frame and expression of the fusedpolypeptide is under control of the same promoter(s) and terminator. Thehybrid polypeptides may comprise a combination of partial or completepolypeptide sequences obtained from at least two different polypeptideswherein one or more may be heterologous to the mutant cell.

An isolated nucleic acid sequence encoding a heterologous polypeptide ofinterest may be manipulated in a variety of ways to provide forexpression of the polypeptide. Expression will be understood to includeany step involved in the production of the polypeptide including, butnot limited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.Manipulation of the nucleic acid sequence prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleic acid sequences utilizing cloningmethods are well known in the art.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, isolated from a naturally occurringgene or modified to contain segments of nucleic acid that are combinedand juxtaposed in a manner which would not otherwise exist in nature.The term nucleic acid construct is synonymous with the term expressioncassette when the nucleic acid construct contains all the controlsequences required for expression of a coding sequence. The term “codingsequence” as defined herein is a sequence that is transcribed into mRNAand translated into a polypeptide. The boundaries of the coding sequenceare generally determined by the ATG start codon located just upstream ofthe open reading frame at the 5′ end of the mRNA and a transcriptionterminator sequence located just downstream of the open reading frame atthe 3′ end of the mRNA. A coding sequence can include, but is notlimited to, genomic, cDNA, RNA, semisynthetic, synthetic, recombinant,or any combinations thereof. The coding sequence of the nucleic acidconstruct described herein may be the nucleotide sequence of theinvention encoding a polypeptide with oxaloacetate activity (as definedin the section above entitled “Isolated Nucleic Acid Sequences EncodingPolypeptides Having Oxaloacetate Hydrolase Activity) in which case thenucleic acid construct is used for producing a polypeptide withoxaloacetate activity as defined in said section or other manipulationinvolving said nucleic acid sequence; or the coding sequence may be oneencoding a heterologous polypeptide to be produced in a mutant cell ofthe invention.

The term “control sequences” is defined herein to include all componentsthat are necessary or advantageous for the expression of a heterologouspolypeptide. Each control sequence may be native or foreign to thenucleic acid sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a heterologous polypeptide. The term “operably linked” isdefined herein as a configuration in which a control sequence isappropriately placed at a position relative to the coding sequence ofthe DNA sequence such that the control sequence directs the productionof a heterologous polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence that is recognized by a cell, in particular a filamentousfungal cell for expression of the nucleic acid sequence. The promotersequence contains transcriptional control sequences that mediate theexpression of the heterologous polypeptide. The promoter may be anynucleic acid sequence that shows transcriptional activity in the cellincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a filamentous fungal cell in the methods ofthe present invention are promoters obtained from the genes encodingAspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Aspergillus oryzae acetamidase (amdS), Fusarium oxysporumtrypsin-like protease (U.S. Pat. No. 4,288,627), and mutant, truncated,and hybrid promoters thereof. Particularly preferred promoters are theNA2-tpi (a hybrid of the promoters from the genes encoding Aspergillusniger neutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), glucoamylase, and TAKA amylase promoters.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by the host cell in question toterminate transcription. The terminator sequence is operably linked tothe 3′ terminus of the nucleic acid sequence encoding the heterologouspolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators functional in filamentous fungal cells areobtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthetase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporumtrypsin-like protease.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA that is important for translation by thecell. The leader sequence is operably linked to the 5′ terminus of thenucleic acid sequence encoding the heterologous polypeptide. Any leadersequence that is functional in the cell may be used in the presentinvention.

Preferred leaders are obtained from the genes encoding Aspergillusoryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence and,when transcribed, is recognized by a cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the cell may be used in the present invention.

Preferred polyadenylation sequences functional in filamentous fungalcells are obtained from the genes encoding Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, and Aspergillus niger alpha-glucosidase.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of theheterologous polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. The foreign signalpeptide coding region may be required where the coding sequence does notnaturally contain a signal peptide coding region. Alternatively, theforeign signal peptide coding region may simply replace the naturalsignal peptide coding region in order to enhance secretion of thepolypeptide. The signal peptide coding region may be obtained from aglucoamylase or an amylase gene from an Aspergillus species, or a lipaseor proteinase gene from a Rhizomucor species. However, any signalpeptide coding region that directs the expressed heterologouspolypeptide into the secretory pathway of a cell may be used in thepresent invention.

An effective signal peptide coding region in a filamentous fungal cellis the signal peptide coding region obtained from the genes encodingAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Rhizomucor miehei aspartic proteinase gene, and Humicola lanuginosacellulase.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature, active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes encoding Rhizomucor miehei aspartic proteinase and Myceliophthorathermophila laccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of the polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

The nucleic acid constructs may also comprise one or more nucleic acidsequences that encode one or more factors that are advantageous fordirecting the expression of the heterologous polypeptide, e.g., atranscriptional activator (e.g., a trans-acting factor), chaperone, andprocessing protease. Any factor that is functional in the host cell, inparticular in a filamentous fungal cell may be used in the presentinvention. The nucleic acids encoding one or more of these factors arenot necessarily in tandem with the nucleic acid sequence encoding theheterologous polypeptide.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the heterologous polypeptide relative tothe growth of the cell. Examples of regulatory systems are those thatcause the expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. The TAKA alpha-amylase promoter, Aspergillus nigerglucoamylase promoter, and Aspergillus oryzae glucoamylase promoter maybe used as regulatory sequences in filamentous fungal cells. Otherexamples of regulatory sequences are those that allow for geneamplification, e.g., the metallothionein genes which are amplified withheavy metals. In these cases, the nucleic acid sequence encoding theheterologous polypeptide would be operably linked with the regulatorysequence.

The various nucleic acid and control sequences described above may bejoined together to produce a recombinant expression vector that mayinclude one or more convenient restriction sites to allow for insertionor substitution of the nucleic acid sequence encoding the heterologouspolypeptide at such sites. Alternatively, the nucleic acid sequenceencoding the heterologous polypeptide may be expressed by inserting thesequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression, and possibly secretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence encodingthe heterologous polypeptide. The choice of the vector will typicallydepend on the compatibility of the vector with the cell into which thevector is to be introduced. The vector may be a linear or closedcircular plasmid. The vector may be an autonomously replicating vector,i.e., a vector that exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, an extrachromosomal element, a minichromosome, or an artificialchromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one that, whenintroduced into the cell, is integrated into the genome and replicatedtogether with the chromosome(s) into which it has been integrated. Thevector system may be a single vector or plasmid or two or more vectorsor plasmids that together contain the total DNA to be introduced intothe genome of the cell, or a transposon.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in a filamentous fungal cell are the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits stableintegration of the vector into a cell genome or autonomous replicationof the vector in the cell independent of the genome of the cell.

“Introduction” means introducing a vector comprising the nucleic acidsequence into a cell so that the vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector. Integrationis generally considered to be an advantage as the nucleic acid sequenceis more likely to be stably maintained in the cell. Integration of thevector into the chromosome occurs by homologous recombination,non-homologous recombination, or transposition.

The introduction of an expression vector into a cell may involve aprocess consisting of protoplast formation, transformation of theprotoplasts, and regeneration of the cell wall in a manner known per se.Suitable procedures for transformation of Aspergillus host cells aredescribed in EP 238 023 and Yelton et al., Proceedings of the NationalAcademy of Sciences U.S.A. 81: 1470–1474 (1984). A suitable method oftransforming Fusarium species is described by Malardier et al., Gene 78:147–156 (1989) or in WO 96/00787.

For integration into the genome of a cell, the vector may rely on thenucleic acid sequence encoding the heterologous polypeptide or any otherelement of the vector for stable integration of the vector into thegenome by homologous or nonhomologous recombination. Alternatively, thevector may contain additional nucleic acid sequences for directingintegration by homologous recombination into the genome of the cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the genome at a precise location(s) in the chromosome(s). Toincrease the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequences that are homologous with the target sequence in thegenome of the cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in thefilamentous fungal cell in question.

It will be understood that the methods of the present invention are notlimited to a particular order for obtaining the mutant cell. Themodification of a gene involved in the production of a polypeptide withoxaloacetate hydrolase activity may be introduced into the parent cellat any step in the construction of the cell for the production of aheterologous polypeptide. It is preferable that the cell has alreadybeen made oxaloacetate hydrolase-deficient using the methods of thepresent invention prior to the introduction of a gene encoding aheterologous polypeptide.

The procedures used to ligate the elements described herein to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatus, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.(1989)).

The present invention also relates to methods for obtaining oxaloacetatehydrolase-deficient mutant cells, in particular filamentous fungalmutant cells which comprise (a) introducing into a parent cell, inparticular a parent filamentous fungal cell a first nucleic acidsequence comprising a modification of at least one of the genes involvedin the production of a oxaloacetate hydrolase and a second nucleic acidsequence encoding a heterologous polypeptide; and (b) identifying themutant from step (a) comprising the modified nucleic acid sequence,wherein the mutant cell produces less of the oxaloacetate hydrolase thanthe parent cell of the mutant cell when cultured under the sameconditions.

The present invention also relates to oxaloacetate hydrolase-deficientmutants of cells, in particular filamentous fungal cells for producing aheterologous polypeptide which comprise a first nucleic acid sequencecomprising a modification of at least one of the genes involved in theproduction of a polypeptide with oxaloacetate hydrolase activity and asecond nucleic acid sequence encoding the heterologous polypeptide,wherein the mutant produces less of the polypeptide with oxaloacetatehydrolase activity than the parent cell of the mutant cell when culturedunder the same conditions.

In another aspect of the present invention, the mutant cell mayadditionally contain modifications of one or more nucleic acid sequenceswhich encode proteins that may be detrimental to the production,recovery, and/or application of the desired product such as theheterologous polypeptide of interest. The modification reduces oreliminates expression of the one or more third nucleic acid sequencesresulting in a mutant cell with a modified third nucleic acid sequencewhich may produce more of the heterologous polypeptide than the mutantcell without the modification of the third nucleic acid sequence whencultured under the same conditions. The third nucleic acid sequence mayencode any protein or enzyme. For example, the enzyme may be anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, a pectinolytic enzyme,peroxidase, phospholipase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, and xylanase. The third nucleicacid sequence preferably encodes a proteolytic enzyme, e.g., anaminopeptidase, a carboxypeptidase, or a protease.

In a further aspect, the present invention relates to a protein productessentially free from oxaloacetate hydrolase activity which is producedby a method of the present invention.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and host cells containing the nucleicacid sequence of SEQ ID NO: 1, subsequences or homologues thereof (asdefined in the section entitled “Isolated Nucleic Acid SequencesEncoding Polypeptides Having Oxaloacetate Hydrolase Activity”), forexpression of the sequences. The constructs and vectors may beconstructed as described herein. The host cell may be any cell suitablefor the expression the nucleic acid sequence, in particular any of thecells mentioned in the section entitled “Isolated Nucleic Acid SequencesEncoding Polypeptides Having Oxaloacetate Hydrolase Activity” as asource for the nucleic acid sequences of the invention. In particular,the host cell is a filamentous fungal cell, such as a cell ofAspergillus, such as A. niger or A. oryzae or a cell of Fusarium.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., Proceedings of the National Academy of Sciences U.S.A.81: 1470–1474 (1984). Suitable methods for transforming Fusarium speciesare described by Malardier et al., 1989, Gene 78: 147–156 and WO96/00787. Yeast may be transformed using the procedures described byBecker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guideto Yeast Genetics and Molecular Biology, Methods in Enzymology 194:182–187, Academic Press, Inc., New York; Ito et al., Journal ofBacteriology 153: 163 (1983); and Hinnen et al., Proceedings of theNational Academy of Sciences U.S.A. 75: 1920 (1978).

Methods of Production

The present invention also relates to methods for producing apolypeptide encoded by a nucleotide sequence of the invention comprising(a) cultivating a host cell harboring a nucleotide sequence of theinvention under conditions suitable for production of the polypeptide;and (b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequence having atleast one mutation in the mature polypeptide coding region of SEQ ID NO:1, wherein the mutant nucleic acid sequence encodes a polypeptide whichconsists of amino acids 1 to 341 of SEQ ID NO: 2, and (b) recovering thepolypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Jansonand Lars Ryden, editors, VCH Publishers, New York (1989)).

Uses

The present invention is also directed to methods of using thepolypeptides having oxaloacetate hydrolase activity.

The polypeptides of the present invention may be used as a diagnosticenzyme, e.g., for the detection of oxalic acid in food or otherproducts.

The nucleotide sequences of the present invention may be used formodification of the production of oxaloacetate hydrolase and thus oxalicby a cell, such as a microbial cell normally producing the hydrolase. Inparticular, the nucleotide sequences may be used to reduce or eliminateoxaloacetate hydrolase and thus oxalic acid production of the cell inquestion.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains:

-   Aspergillus niger BO 1 (DSM 12665)-   Escherichia coli DH 5α D. M. Woodcock et al., Nucleic Acids Res. 17:    3469–3478 (1989)    Media and Assays-   Buffer A: 50 mM Tris/HCl pH 7.5, 2 mM MnCl₂, 20 mM DTT, 5% sucrose.-   Oxaloacetate hydrolase activity assay: 1000 ml 0.1 M MOPS, pH 7.5, 2    mM MnCl₂, 25 ml 40 mM oxaloacetate, 5 to 100 ml sample. The    absorbance was measured at 255 nm. The activity was determined from    the rate of decrease in absorbance and the absorption coefficient of    oxaloacetate (1.1 mM⁻¹ cm⁻¹, Lenz et al, Partial purification and    some properties of oxalacetase from Aspergillus niger, Eur. J.    Biochem. 65: 225–236 (1976)). The assay was carried out at 30° C.    Protein assay: Protein concentrations were measured by using the    BioRad (Hercules, Calif., U.S.A.) Bio-Rad Protein Assay) cat. No.    500-0006 following the manufacturer's instructions and with bovine    serum albumine as a standard.

Example 1

Purification of the Oxaloacetate Hydrolase (EC 3.7.1.1)

Aspergillus niger BO1 was fermented in shake flasks at 30° C. in themedium: sucrose 20 g/L, NaNO₃ 15 g/L, KH₂PO₄ 1.5 g/L, MgSO₄, 7H₂O 1 g/L,NaCl 1 g/L, CaCl₂, 2H₂O 0.1 g/L, trace solution 0.5 ml/L. Tracesolution: ZnSO₄, 7H₂O 14.3 g/L, CuSO₄, 5H₂O 2.5 g/L, NiCl₂, 6H₂O 0.5g/L, FeSO₄, 7H₂O 13.8 g/L, MnCl₂ 6 g/L. The pH was 2.5 until a biomassconcentration of about 0.5 g/L was reached. Then the pH was shifted to 6by addition of 2 M NaOH and the cells were grown until the biomassconcentration reached about 5 g/L. The cells were harvested byfiltration, washed with 0.9% (w/v) NaCl and then frozen in liquidnitrogen. The frozen cells were disrupted in a morter under liquidnitrogen and then suspended in buffer A. The suspension was centrifuged(15 min, 40.000×g, 4° C.), and the supernatant was isolated.Ammoniumsulphate was added to 45% saturation (277 g/l), and the formedsuspension was centrifuged (15 min, 40.000×g, 4° C.), and thesupernatant was discharged. The pellet was dissolved in buffer A andfiltered through a 0.45 micro-m filter.

Ammonium sulfate was added to the sample to a conductivity of 50 mS/cm.The sample was then applied onto a Phenyl Sepharose High Performancecolumn (Amersham Pharmacia Biotech, Uppsala) equilibrated with 0.5 Mammonuim sulfate dissolved in buffer A, following the manufacturer'sinstructions. The column was washed with the same buffer and then elutedusing a linear salt gradient from 0.5 M Ammonium sulfate dissolved inbuffer A to pure buffer A.

Fractions having oxaloacetate hydrolase activity were pooled. Thissolution was diluted with 2 mM MnCl₂, 20 mM DTT, 5% sucrose to aconductivity of 4 mS/cm and then applied to a Q sepharose HighPerformance column (Amersham Pharmacia Biotech, Uppsala) equilibratedwith buffer A. The column was washed with buffer A and then eluted usinga linear salt gradient from 0 M to 0.5 M NaCl dissolved in buffer A.Fractions having oxaloacetate hydrolase activity were pooled.

The samples were applied to PD-10 columns (Amersham Pharmacia Biotech,Uppsala) equilibrated with 10 mM NaH₂PO₄ pH 7.2, 0.1 mM MnCl₂, 5%sucrose, 10 mM DTT and eluted with the same buffer. The samples wereapplied to a Econo-Pac HTP column (BioRad, Hercules, Calif., U.S.A.)that was equilibrated with 10 mM NaH₂PO₄ pH 7.2, 0.1 mM MnCl₂, 5%sucrose, 10 mM DTT. The column was washed with 10 mM NaH₂PO₄ pH 6.8, 0.1mM MnCl₂, 5% sucrose, 10 mM DTT and then eluted with a linear gradientfrom 10 mM NaH₂PO₄ pH 6.8, 0.1 mM MnCl₂, 5% sucrose, 10 mM DTT to 400 mMNaH₂PO₄ pH 6.8, 0.1 mM MnCl₂, 5% sucrose, 10 mM DTT. The samples havingoxaloacetate hydrolase activity were used for SDS-PAGE.

Example 2

Protein Sequence Determination of Fragments of the OxaloacetateHydrolase

The purified oxaloacetate hydrolase of example 1 was subjected toSDS-PAGE gel electrophoresis on a 12.5% gel using standard methods(Laemmli, U. K., Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4, Nature 227: 680–685 (1970)). The proteinswere blotted onto a PVDF membrane by electroblotting, and the membranewas stained with Coomassie brilliant blue R-250 as described by Ploughet al (Plough M, Jensen A. L. and Barkholt V., Anal. Biochem. 181: 33–39(1989)). 4 protein bands in the range of an apparent molecular weight of38 to 40 kD were excised and applied for amino terminal sequencing usinga Procise −494 protein sequencer from PerkinElmer—Applied Biosystems,Norwalk, Conn., U.S.A. following the manufacturer's instructions. Thefollowing sequences were obtained:

1. MKVDTPDSASTISMTN (SEQ ID NO: 3) 2. TNTITITVEQDGIYE (SEQ ID NO: 4) 3.VEQDGIYEIN (SEQ ID NO: 5) 4. GARQEPVVNLNMVTG. (SEQ ID NO: 6)

Example 3

Cloning of the Gene Encoding the Oxaloacetate Hydrolase

The protein sequences determined in example 2 were used for databasesearches, and an Aspergillus niger EST sequence was retrieved (EMBL,T82752, AN752). This EST encoded a protein sequence that could bealigned to the four sequences from example 2 with 100% identity. The ESTsequence was used to design the following PCR primers:

(SEQ ID NO: 7) Oxalac EST 5′ AAA GTT GAT ACC CCC GAT TCT 3′ sense: (SEQID NO: 8) Oxalac EST 5′ ATG GCA ATA CGG GGA CAG ACC 3′. antisense:

These primers were used to amplify DNA from A. niger BO 1 preparedessentially as described by Leach et. al. (J. Leach, D. B. Finkelsteinand J. A. Rambosek, Fungal Gent. Newsl. 33: 32–33 (1986)) using amplitaqtaq polymerase from PerkinElmer (Norwalk, Conn., U.S.A.) following themanufacturer's instructions. The PCR reaction was run in an MJ PCT 150capillary PCR cycler (M. J. research, Watertown, Mass., U.S.A. in avolume of 10 microliters running 30 cycles with a denaturationtemperature of 94° C. for 10 sec, an annealing temperature of 55° C. for10 sec and an elongation temperature of 72° C. for 30 sec with atemperature gradient from denaturation to annealing of −0.5° C./sec.

The obtained 219 bp fragment was ligated into the pCR II vector(Invitrogen, Carlsbad, Calif., U.S.A.) following the manufacturer'sinstructions and transformed into Escherichia coli DH 5α as described bySambrook (J. Sambrook et al., Molecular Cloning, A Laboratory Manual,second edition, Cold Spring Harbor Laboratory Press (1989)). A clonecontaining an insert of the expected size was sequenced using theprimers:

(SEQ ID NO: 9) M13 reverse 5′ CAG GAA ACA GCT ATG AC 3′. primer: (SEQ IDNO: 10) M13 Forward (-20) 5′ GTA AAA CGA CGG CCA G 3′. primer:

The DNA sequence analysis was made using an ABI PRISM 377 DNA Sequencertogether with the ABI PRISM BigDye Terminator Cycle Sequencing ReadyReaction Kit (PerkinElmer Applied Biosystems, Foster city, Calif.,U.S.A.) following the manufacturer's recommendations. The sequencesconfirmed the fragment to be the desired sequence.

A restriction map was established by Southern blot analysis using the219 bp insert as a probe following the protocols of Sambrook et al. Fromthis restriction map it was concluded that the gene was located on a 5kb Bgl II fragment.

The gene was then cloned by inverse PCR: Genomic DNA from A. niger BO 1was digested with Bgl II, and fragments ranging from 4 kb to 6 kb wererecovered from an agarose gel. The DNA was ligated using T4 DNA ligaseat 16° C. This ligation mixture was used as template in a PCR reactionusing the following primers:

(SEQ ID NO: 11) OXEST1 5′ GAC GGT CTG TCG CCG TAT TGC 3′ (SEQ ID NO: 12)OXEST2 5′ GGA AGC AGA ATC GGG GGT ATC 3′.

The Expand high fidelity polymerase (Boehringer Mannheim, Mannheim,Germany) was used for the amplification following the manufacturer'srecommendations. The MgCl₂ concentration in the reaction was 1 mM.Otherwise the conditions were as above. A 5 kb fragment was generated.This fragment was digested with Bgl II, and the digested fragment wasligated to pUC 19 (C. Yanisch-Perron et al., Gene 33: 103–119 (1985))digested with BamH I and Hinc II. The ligation mixture was transformedinto E. coli DH 5α. Colonies having insert sizes of 1.3 kb and 3.7 kbwere found. One colony of each class were sequenced using the M13reverse primer and the M13 Forward (−20) primer. In this way the Bgl IIneighboring sequences were determined, and the following primers weredesigned to PCR amplify the 5 kb Bgl II fragment:

Flank 1: 5′ GCG GCC GCG CGC CAA TAA CGT CCG ATT C 3′ (SEQ ID NO: 13)Flank 2: 5′ GCG GCC GCC TAC AAA TAC ATT GAC CTC CC 3′. (SEQ ID NO: 14)

These primers were used to amplify genomic A. niger BO 1 DNA using thesame PCR conditions as for the inverse PCR. The generated 5 kb fragmentwas ligated into pCR II to form pHP I. The oxaloacetate hydrolase genewas sequenced using pHP I as template with the following primers:

Oxalac EST sense: 5′ AAA GTT GAT ACC CCC GAT TCT 3′ (SEQ ID NO: 15)Oxalac EST antisense: 5′ ATG GCA ATA CGG CGA CAG ACC 3′ (SEQ ID NO: 16)OXEST 1: 5′ GAC GGT CTG TCG CCG TAT TGC 3′ (SEQ ID NO: 17) OXEST 2: 5′GGA AGC AGA ATC GGG GGT ATC 3′ (SEQ ID NO: 18) OXEST 3: 5′ GCC GGA GTCGCG GGA TTC CAC 3′ (SEQ ID NO: 19) OXEST 4: 5′ GGC GGA CTA TGA TTT GTGCC 3′ (SEQ ID NO: 20) OX5: 5′ TGA TGG TCG CCC GTT CCG TT 3′ (SEQ ID NO:21) OX6: 5′ TGC CAT TCA ATT TTC TTG GCC 3′ (SEQ ID NO: 22) OX7: 5′ TGATCT TCG ATG TGG AAT CCC 3′ (SEQ ID NO: 23) OX8: 5′ GAT GGC GTC GAT TGACCA TTT 3′ (SEQ ID NO: 24) OX9: 5′ GGA GAT GGG TTT GCT AAT GGT GTT 3′(SEQ ID NO: 25) OX10: 5′ TTA GCA AAC CCA TCT CCA CC 3′ (SEQ ID NO: 26)OX11: 5′ CGA ATT ACT GGT CAT TAG CCC 3′ (SEQ ID NO: 27) OX12: 5′ CGA GAGAAG TAT TCT AGA CCC 3′ (SEQ ID NO: 28) OX13: 5′ TGA CTG TCG ATC AGG GTGTT 3′ (SEQ ID NO: 29) OX14: 5′ GTG TGC GGA TTG ATG GAC TC 3′ (SEQ ID NO:30) OX15: 5′ CAA CCC AAC TCA ACA ACT CT 3′ (SEQ ID NO: 31) FLANKE1: 5′GCG GCC GCG CGC CAA TAA CGT CCG ATT C 3′ (SEQ ID NO: 32) FLANKE2: 5′ GCGGCC GCC TAC AAA TAC ATT GAC CTC CC 3′ (SEQ ID NO: 33)

The DNA sequence is shown in SEQ ID NO: 1. The sequence was analyzed forcoding sequence and for the presence of introns using the computersoftware Netgene 2 (S. M. Hebsgaard, P. G. Korning, N. Tolstrup, J.Engelbrecht, P. Rouze, S. Brunak (Nucleic Acids Research 24: 3439–3452(1996)) suggested the existence of 3 exons (see annotations to SEQ IDNO: 1). The 341 residue protein sequence deduced from these 3 introns isshown in SEQ ID NO: 2.

Data base searches using this protein sequence (oah) as the querysequence revealed sequence homology to Isocitrate lyase from Aspergillusnidulans (Swiss prot P28298) and Neurospora crassa (Swiss prot P28299)as well as carboxyvinyl-carboxyphosphonate phosphorylmutase fromStreptomyces hygroscopicus (Swiss prot P11435) and a hypotheticalprotein from Bacillus subtilis (Swiss prot P54528).

Oah P28298 P28299 P11435 P54528 oah 100.0 20.2 21.0 29.7 29.1 P2829820.2 100.0 74.3 17.6 19.3 P28299 21.0 74.3 100.0 18.1 18.6 P11435 29.717.6 18.1 100.0 36.5 P54528 29.1 19.3 18.6 36.5 100.0

A strain of E. coli harboring the plasmid pHP1 was deposited as DSM12660.

Example 4

Disruption of the Oxaloacetate Hydrolase Gene

pHP I was digested with Nru I and BstE II and the 6.6 kb fragment wasisolated. A plasmid harboring the Aspergillus niger pyrG gene, pJRoy 10(FIG. 1), was digested with Nco I, and the 5′ recessive ends were thenfilled in with the Klenow fragment of DNA polymerase I, and thendigested with BstE II. The 2948 bp fragment was isolated and ligated tothe pHP I fragment. The ligation mixture was used to transform E. coliDH5α. A colony harboring the desired plasmid was identified, and theplasmid was termed pHP 3 (FIG. 2). A strain of E. coli harboring theplasmid pHP3 was deposited as DSM 12661.

The pyrG deficient A. niger BO 1 derivative JRoy3 (BO1 was rendered pyrGnegative by transforming with a deletion fragment of the pyrG generegion and selecting on FOA) was transformed with the 5 kb EcoR I-Not Ifragment of pHP 3 using the method described in the EP patent EP 0 531372 B1. 275 transformants were reisolated twice and then grown up in 96well microtiter dishes at 34° C., pH 6.0 for 48 hr in the medium glucose20 g/L, NaNO₃ 15 g/L, KH₂PO₄ 1.5 g/L, MgSO₄, 7H₂O 1 g/L, NaCl 1 g/L,CaCl₂, 2H₂O 0.1 g/L, tracer solution 0.5 ml/L. Tracer solution: ZnSO₄,7H₂O 14.3 g/L, CuSO₄, 5H₂O 2.5 g/L, NiCl₂, 6H₂O 0.5 g/L, FeSO₄, 7H₂O13.8 g/L, MnCl₂ 6 g/L. The supernatants were assayed for oxalate usingthe Sigma (St. Louis, Mo., U.S.A.) oxalate (cat no 591-C) kit. Eighttransformants were found not to produce oxalate. 6 of those weresubjected to Southern blot analysis together with 2 oxalate producingtransformants, BO 1 and JRoy 3 as positive controls. The 219 bp EST PCRfragment described in example 3 was used as probe. A separate blot ofthe same samples was probed with the Nco I-BstE II fragment of pJRoy 10.The Southern blots revealed that the 6 oxalate negative strains weredisrupted in the oxaloacetate gene, whereas the positive controlscontained intact oxaloacetate genes.

Example 5

Fermentation of an Oxalate Negative Strain

One of the oxalate negative strains was grown in a batch fermenterequipped with stirring, temperature control, pH control and aeration.The medium was glucose 16 g/L, (NH4)₂SO₄ 7.5 g/L, KH₂PO₄ 1.5 g/L, MgSO₄,7H₂O 1 g/L, NaCl 1 g/L, CaCl₂, 2H₂O 0.1 g/L, trace solution 0.5 ml/L.Trace solution: ZnSO₄, 7H₂O 14.3 g/L, CuSO₄, 5H₂O 2.5 g/L, NiCl₂, 6H₂O0.5 g/L, FeSO₄, 7H₂O 13.8 g/L, MnCl₂ 6 g/L. The pH was 2.5 until thebiomass had reached a concentration of 0.5 g/L. At this point the pH wasshifted to pH 6.0. The fermentation broth was analyzed for oxalate,citrate, pyruvate, succinate, acetate, glycerol, ethanol and glucose(Nissen, T. L., U. Schulze, J. Nielsen, and J. Villadsen, Fluxdistributions in anaerobic, glucose-limited continuous cultures ofSaccharomyces cerevisiae, Microbiol. 143: 203–218 (1997)). Ofbi-products only citrate, pyruvate, succinate and glycerol could bedetected and the maximum concentrations of these bi-products were 0.74g/L, 0.18 g/L, 0.14 g/L and 0.13 g/L respectively. The biomass yield onglucose was 0.58 g/g and the specific growth rate was 0.23 h⁻¹.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the DSMZ-Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig,Germany, and given the following accession numbers:

Depositor's ref: Accession Number Date of Deposit NN049454 DSM 12660Feb. 3, 1999 NN049455 DSM 12661 Feb. 3, 1999 NN049047 DSM 12665 Feb. 3,1999

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for producing citric acid, comprising (a) culturing a mutantof a parent cell, wherein the mutant produces less oxaloacetatehydrolase than the parent cell, wherein the oxaloacetate hydrolase (i)has an amino acid sequence that has at least 90% identity with aminoacids 1-341 of SEQ ID NO:2; (ii) is encoded by a nucleic acid sequencehaving at least 90% homology with the cDNA sequence of SEQ ID NO: 1;and/or (iii) is encoded by a nucleic acid sequence which hybridizesunder high stringency conditions with the nucleic acid sequence of SEQID NO: 1, the cDNA sequence of SEQ ID NO: 1, the complete complementarystrand of SEQ ID NO: 1, and/or the complete complementary strand of thecDNA sequence of SEQ ID NO: 1, wherein the high stringency conditionsare defined as prehybridization and hybridization at 42° C. in 5×SSPE,0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and50% formamide, followed by washing three times for 15 minutes using2×SSC, 0.2% SDS at 65° C.; and (b) recovering the citric acid.
 2. Themethod of claim 1, wherein the oxaloacetate hydrolase has an amino acidsequence that has at least 90% identity with amino acids 1–341 of SEQ IDNO:
 2. 3. The method of claim 2, wherein the oxaloacetate hydrolase hasan amino acid sequence that has at least 95% identity with amino acids1–341 of SEQ ID NO:
 2. 4. The method of claim 3, wherein theoxaloacetate hydrolase has an amino acid sequence that has at least 97%identity with amino acids 1–341 of SEQ ID NO:
 2. 5. The method of claim1, wherein the oxaloacetate hydrolase has an amino acid sequence whichcomprises SEQ ID NO: 2 or a fragment of SEQ ID NO: 2 that hasoxaloacetate hydrolase activity.
 6. The method of claim 1, wherein theoxaloacetate hydrolase has the amino acid sequence of SEQ IDNO:
 2. 7.The method of claim 1, wherein the parent cell comprises the nucleicacid sequence of SEQ IDNO:
 1. 8. The method of claim 1, wherein theparent cell comprises the cDNA sequence of SEQ ID NO:
 1. 9. The methodof claim 1, wherein the oxaloacetate hydrolase is encoded by a nucleicacid sequence that hybridizes under said high stringency conditions withthe nucleic acid sequence of SEQ ID NO: 1, the cDNA sequence of SEQ IDNO: 1, the complete complementary strand of SEQ ID NO: 1, and/or thecomplete complementary strand of the cDNA sequence of SEQ ID NO:
 1. 10.The method of claim 1, wherein the parent cell is a filamentous fungalcell.
 11. The method of claim 10, wherein the parent cell is a cell ofAcremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora,Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia,Tolypocladium, or Trichoderma strain.
 12. The method of claim 11,wherein the parent cell is a cell of Aspergillus.
 13. The method ofclaim 12, wherein the parent cell is a cell of A. niger.
 14. The methodof claim 1, wherein the mutant produces at least about 25% less of theoxaloacetate hydrolase than the parent cell when cultured underidentical conditions.
 15. The method of claim 14, wherein the mutantproduces no oxaloacetate hydrolase.